Stephen C. Richter

Histamine-releasing and antimicrobial peptides from the skin secretions of the Dusky Gopher frog, Rana sevosa

Seven novel peptides were isolated from the skin secretions of the North American dusky gopher frog, Rana sevosa, on the basis of antimicrobial activity and histamine release from rat peritoneal mast cells. The peptides were purified to homogeneity using HPLC and characterized by electrospray ion-trap mass spectrometry, MALDI-TOF mass spectrometry and Edman sequencing. Bioinformatic analysis of primary structures revealed that the novel peptides could be assigned to four established families of ranid frog antimicrobial peptides, namely esculentin-1, esculentin-2, brevinin-1 and ranatuerin-2. The peptides were named in accordance with accepted terminology as ranatuerin 2SEa, etc., reflecting the peptide family name, the species of origin (SE for sevosa) and the isotype (a). Of major interest was the fact that brevinin 1SE displayed significant structural similarity to ponericin W5, an antibacterial venom peptide from the ant, Pachyconyla goeldii. This is a further example of amphibian skin defensive peptides showing striking structural similarities to peptides from insects. These data may shed some light on the functional biological relevance of defensive peptides that possess both antimicrobial and histamine-releasing activities.

Peptide Tyrosine Arginine, a potent immunomodulatory peptide isolated and structurally characterized from the skin secretions of the dusky gopher frog, Rana sevosa

An octadecapeptide was isolated from the skin secretions of the dusky gopher frog (Rana sevosa) on the basis of histamine release from rat peritoneal mast cells. This peptide was purified to homogeneity by HPLC and found to have the following primary structure, YLKGCWTKSYPPKPCFSR, using both Edman degradation chemistry and peptide sequencing using high-resolution mass spectrometry (Q-TOF MS). The peptide, named peptide Tyrosine Arginine (pYR) shares 77.8% homology with peptide Leucine Arginine (pLR). The effects of the natural amidated peptide, non-amidated peptide and C-loop region of pYR on granulopoiesis and neutrophil apoptosis were investigated. All three analogues inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, the mature neutrophil. Thus, pYR is a novel member of an important and emerging new class of amphibian peptides with hemopoietic actions.

The Significant Surface-Water Connectivity of “Geographically Isolated Wetlands"

We evaluated the current literature, coupled with our collective research expertise, on surface-water connectivity of wetlands considered to be “geographically isolated” (sensu Tiner Wetlands 23:494–516, 2003a) to critically assess the scientific foundation of grouping wetlands based on the singular condition of being surrounded by uplands. The most recent research on wetlands considered to be “geographically isolated” shows the difficulties in grouping an ecological resource that does not reliably indicate lack of surface water connectivity in order to meet legal, regulatory, or scientific needs. Additionally, the practice of identifying “geographically isolated wetlands” based on distance from a stream can result in gross overestimates of the number of wetlands lacking ecologically important surface-water connections. Our findings do not support use of the overly simplistic label of “geographically isolated wetlands”. Wetlands surrounded by uplands vary in function and surface-water connections based on wetland landscape setting, context, climate, and geographic region and should be evaluated as such. We found that the “geographically isolated” grouping does not reflect our understanding of the hydrologic variability of these wetlands and hence does not benefit conservation of the Nation’s diverse wetland resources. Therefore, we strongly discourage use of categorizations that provide overly simplistic views of surface-water connectivity of wetlands fully embedded in upland landscapes.

Temporal trends in genetic data and effective population size support efficacy of management practices in critically endangered dusky gopher frogs ( Lithobates sevosus )

Abstract Monitoring temporal changes in population genetic diversity and effective population size can provide vital information on future viability. The dusky gopher frog, Lithobates sevosus, is a critically endangered species found only in coastal Mississippi, with low genetic variability as a consequence of isolation and population size reduction. Conservation management practices have been implemented, but their efficacy has not been addressed. We genotyped individuals collected 1997–2014 to determine temporal trends in population genetic variation, structure, and effective size. Observed and expected heterozygosity and allelic richness revealed temporally stable, but low, levels of genetic variation. Positive levels of inbreeding were found in each year. There was weak genetic structure among years, which can be attributed to increased effects of genetic drift and inbreeding in small populations. L. sevosus exhibited an increase in effective population size, and currently has an estimated effective size of 33.0–58.6 individuals, which is approximately half the census size. This large ratio could possibly be explained by genetic compensation. We found that management practices have been effective at maintaining and improving effective size and genetic diversity, but that additional strategies need to be implemented to enhance viability of the species.

Digital Daisy: An Inquiry-Based Approach to Investigating Floral Morphology and Dissection

Many topics in the secondary science classroom can be difficult to introduce to students in a manner that fully engages them, especially when presented using traditional teaching methods. However, with a little innovation and an emphasis on inquiry, even dry subjects can be presented in an appealing way. The authors developed an inquiry-based exercise that teaches students about flower anatomy and dissection. During this exercise, students investigate the morphology of simple and composite flowers using digital microscopes. Dissection of a simple flower results in a count of floral parts that is used to construct a floral formula. Students use appearance of structures to suggest function of sepals, petals, anthers, pistils, and components. Investigation of composite and bilabiate flowers introduces concepts of inflorescence and symmetry and asymmetry. Following self-guided inquiry by the students, the teacher uses a digital microscope, computer, and LCD projector to lead discussion of what was observed and to help assimilate important concepts.

Population and Conservation Genetics of Crawfish Frogs, Lithobates areolatus, at Their Northeastern Range Limit

Abstract Crawfish Frogs (Lithobates areolatus) are a North American ranid, considered near threatened globally with populations in decline throughout their range. We studied populations of Crawfish Frogs on local and regional scales at their northeastern range limit to (1) assess the level of genetic diversity within populations, (2) estimate fine-scale genetic structure, and (3) estimate genetic differentiation between populations at the regional level. We used 10 microsatellite loci to genotype frogs collected from three regional sites in Indiana separated by 50–172 km and at one of these sites within a network of three breeding ponds <1 km apart. Heterozygosity estimates revealed high levels of diversity within these populations (mean HO: 0.54–0.67 per site), which is encouraging for future management. The degree of population subdivision was low at the regional level (FST = 0.071 for sites within 172 km). Genetic differentiation was related to geographic distance between sampling sites, as predicted by an isolation-by-distance model. We observed no genetic differentiation between individuals sampled from ponds approximately 250 m apart and slight divergence of individuals from a pond approximately 750 m away. This suggests ponds within 1 km form a genetically distinct single breeding unit composed of multiple subpopulations. Finally, we observed high genetic differentiation between southwest and southeast Indiana sites indicating historical (rather than recent) isolation of these sites. These data will be applied to a regional management plan in an attempt to recover Crawfish Frogs along the northeastern extreme of their range.

Field-based assessment of wetland condition, wetland extent, and the National Wetlands Inventory in Kentucky, USA

The economic and ecological importance of wetlands is well documented, but there are few studies that have assessed wetland condition and extent for the United States. Many states, including Kentucky, have had no statewide field evaluation of wetlands of any kind. The National Wetland Inventory (NWI) is the largest database for mapped wetlands in the United States and the most comprehensive source of wetland information for Kentucky, but its value for determining wetland condition is limited. Therefore, our objectives were to document wetland extent and condition and assess the agreement between the NWI and field-based wetland characteristics in Kentucky. We conducted field and remote-sensing based assessments of 352 wetlands across the state. NWI-mapped and field-assessed wetlands had similar large-scale patterns; however, for individual wetlands, classification often disagreed. Based on our wetland assessment method, wetlands appear to be of moderate condition, although we found differences among basins, dominant vegetation types, and landscape positions and much variation as many sites scored very low and high. Our findings support previous work showing that rapid assessments are valuable for determining wetland condition for ambient monitoring and other applications. Also, our results provide the foundation for future status and trends studies and suggest an urgent need to update the NWI in Kentucky and elsewhere. We suggest that the NWI could be improved by using newer technology that increases wetland mapping accuracy and including predictions of wetland condition using the enhanced NWI approach.

Microhabitat conditions influence mesohabitat associations and distribution of larval salamanders in headwater streams

Distribution patterns of stream biota are the result of complex interactions between individuals and their surrounding environment. Determining the spatial scale by which an organism is most influenced is paramount to understanding distribution patterns. Using a multi-scale approach, we investigated factors influencing habitat associations of larval Ambystoma barbouri (streamside salamander) and Eurycea cirrigera (southern two-lined salamander) in three Kentucky headwater streams. We used likelihood ratio G tests to identify associations between species and mesohabitat (i.e., runs, riffles, and pools), and we used microhabitat variables to predict the presence and abundance of salamanders via a priori multiple regression modeling. Ambystoma barbouri presence and abundance were influenced by conditions at micro-scales, which in turn dictated mesohabitat associations. Eurycea cirrigera were also influenced by microhabitat variables, but displayed associations to A. barbouri presence in late spring. Associations of larval salamanders to mesohabitat and microhabitat parameters shifted from early to late spring, likely in response to changes in developmental stage. The multi-scale approach of our study improved our understanding of complex relationships between larval salamanders and their surrounding environment in headwaters, and underscored the importance of (1) research investigating multiple spatial and temporal scales and (2) heterogeneous in-stream habitat to headwater biota.

Cryptic Diversity and Conservation of Gopher Frogs across the Southeastern United States

Identifying cryptic biodiversity is fundamental to evolutionary biology and to conservation efforts. This study investigated range-wide genetic diversity of Gopher Frogs, Lithobates capito, across the southeastern United States coastal plain to determine implications for taxonomy and conservation. We collected data for two mtDNA regions in 21 populations to identify genetic structure across the geographic distribution of the species. Based on population genetic, phylogenetic, and genealogical analyses, we recovered three reciprocally monophyletic mtDNA lineages corresponding to mainland coastal plain populations and two lineages within peninsular Florida. Breakpoints for these lineages did not occur in previously identified hotspots of amphibian phylogeographic breaks and did not follow currently recognized subspecies designations. We recommend these lineages be recognized as separate distinct population segments and be considered separately by the U.S. Fish and Wildlife Service for listing under the Endangered Species Act. Additionally, we propose an evolutionary hotspot for amphibians that deserves further attention.

Fourteen novel microsatellite markers for the gopher frog, Lithobates capito (Amphibia: Ranidae)

We isolated and characterized a total of 14 microsatellite loci from gopher frogs, Lithobates capito. This species is of conservation concern because most populations have gone locally extinct across the geographic distribution. Loci were screened for 21 individuals from a single population in Florida. The number of alleles per locus ranged from 7 to 17, observed heterozygosity ranged from 0.667 to 0.947, and the probability of identity values ranged from 0.011 to 0.077. These new loci provide tools for examining the genetic diversity and population structure of L. capito populations and addressing factors associated with their decline.